Microbiological test methods for the determination of antibacterial compounds, particularly residues of antibiotics such as cephalosporin, penicillin, tetracycline and derivatives thereof and chemotherapeutics such as sulfa's, in fluids such as milk, meat juice, serum and urine have been known for a long time. Examples of such tests have been described in CA 2056581, DE 3613794, EP 0005891, EP 0285792, EP 0611001, GB A 1467439 and U.S. Pat. No. 4,946,777. These descriptions all deal with ready to use tests that make use of a test organism and will give a result by the change indicated by an indicator molecule added to the test system. The principle is that when an antibacterial compound is present in the fluid in a concentration sufficient to inhibit the growth of the test organism the color of the indicator will stay the same, while, when no inhibition occurs, the growth of the test organism is accompanied by the formation of acid or reduced metabolites or other phenomena that will induce an indicator signal.
The test systems mentioned above include a test medium, such as an agar medium, inoculated with a test organism, preferably a strain of Bacillus, Escherichia coli or Streptococcus, and a pH indicator and/or a redox indicator. The test organism and the indicator are introduced into an optionally buffered agar solution wherein nutrients are present and optionally substances to change the sensitivity to certain antimicrobial compounds. The agar solution is allowed to solidify to form the test medium such that the test organisms stay alive but cannot multiply because of low temperature. A suitable test should have the desired sensitivity with regard to the compounds to be tested for.
The test systems currently distributed on the market and/or described in literature all have a certain range in which indicator color changes occur. The width of this range determines how accurate a given test system performs. Many of the known test systems display different results when tests are read after a predetermined amount of time or after the time required for a blank sample to change color. It is desirable to develop test systems whereby the range in which the indicator color change occurs is smaller and/or more easily readable and/or having an improved sensitivity than in known systems whilst simultaneously the stability of the test organism is warranted and whereby there are smaller differences between tests that are determined after a predetermined amount of time versus tests that are determined at the moment that a blank sample changes color.